A forkhead transcription factor contributes to the regulatory differences of pathogenicity in closely related fungal pathogens

Abstract Cryptococcus neoformans and its sister species Cryptococcus deuterogattii are important human fungal pathogens. Despite their phylogenetically close relationship, these two Cryptococcus pathogens are greatly different in their clinical characteristics. However, the determinants underlying the regulatory differences of their pathogenicity remain largely unknown. Here, we show that the forkhead transcription factor Hcm1 promotes infection in C. neoformans but not in C. deuterogattii. Monitoring in vitro and in vivo fitness outcomes of multiple clinical isolates from the two pathogens indicates that Hcm1 mediates pathogenicity in C. neoformans through its key involvement in oxidative stress defense. By comparison, Hcm1 is not critical for antioxidation in C. deuterogattii. Furthermore, we identified SRX1, which encodes the antioxidant sulfiredoxin, as a conserved target of Hcm1 in two Cryptococcus pathogens. Like HCM1, SRX1 had a greater role in antioxidation in C. neoformans than in C. deuterogattii. Significantly, overexpression of SRX1 can largely rescue the defective pathogenicity caused by the absence of Hcm1 in C. neoformans. Conversely, Srx1 is dispensable for virulence in C. deuterogattii. Overall, our findings demonstrate that the difference in the contribution of the antioxidant sulfiredoxin to oxidative stress defense underlies the Hcm1‐mediated regulatory differences of pathogenicity in two closely related pathogens.


INTRODUCTION
Cryptococcus neoformans and Cryptococcus deuterogattii (formerly C. gattii, genotype AFLP6/VGII) represent the most important human fungal pathogens in the phylum Basidiomycota, which contains more than 30,000 known species 1,2 .These pathogenic species are evolutionally close and have nearly identical asexual and sexual life cycles 3,4 .Asexually, C. neoformans and C. deuterogattii mostly proliferate as yeast cells and reproduce by budding.Under favorable conditions, fungi of both species undergo similar sexual differentiation to produce infectious basidiospores, which can be inhaled into the lungs 4 .Both C. neoformans and C. deuterogattii have exceptional capabilities for lung colonization and dissemination from the lung to the brain, causing pneumonia and meningitis [5][6][7] .In addition to their importance as deadly pathogens, with advances in genetics, large-scaled deletion mutant libraries, whole-genome sequencing, and murine models [8][9][10][11][12][13][14] , these two closely related fungi have become excellent models for investigation of the genetic and regulatory bases of fungal pathogenesis, biology, and evolution.
Previous studies have identified multiple common virulence traits in C. neoformans and C. deuterogattii.For instance, both of them can multiply well at typical mammalian body temperatures, which is believed to distinguish them from nonpathogenic Cryptococcus species 15,16 .In addition, they both are able to produce capsules and melanin, which serve as the major virulence factors that facilitate their infectivity 17,18 .Despite these shared virulence traits, C. neoformans and C. deuterogattii are greatly different in their genetic, ecological, clinical, and stress adaptation characteristics [19][20][21] .Different from the sister species C. deuterogattii, which is often associated with plants, C. neoformans is frequently detected in pigeon droppings 14,22 .Furthermore, C. neoformans is one of the most common fungal pathogens causing infections in immunocompromised individuals, whereas C. deuterogattii is the causative agent for an ongoing cryptococcosis outbreak in the Pacific Northwest regions that commonly infects immunocompetent patients [23][24][25] .These differences likely mirror their distinct adaptation abilities to diverse stress from natural niches or hosts.Consistent with this hypothesis, an earlier study indicated a better proliferation of R265, the model strain of C. deuterogattii, in macrophages compared to the C. neoformans reference strain H99.The better proliferation of R265 was demonstrated to be attributed to its more effective defense against reactive oxygen species (ROS) and nitrosative stress within macrophages [26][27][28] .These dissimilarities strongly suggest that there exist regulatory differences of pathogenicity in these phylogenetically close pathogens.However, the determinants involved remain largely unknown.
In this study, we show that the forkhead regulator Hcm1 plays an important regulatory role in the pathogenicity of C. neoformans but not in that of C. deuterogattii.This difference is largely due to the different contributions of Hcm1 to antioxidative defense in two Cryptococcus species.We found that C. neoformans-derived Hcm1, but not its C. deuterogattii homolog, plays a critical role in oxidative stress responses.Chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) and transcriptional assays together with epistasis assessment identified the antioxidant sulfiredoxin gene SRX1 as the conserved target of Hcm1.Likewise, the loss of SRX1 showed a more severe defect in oxidative stress defense in C. neoformans than in C. deuterogattii.Importantly, the overexpression of SRX1 can largely restore the pathogenicity of C. neoformans strain devoid of Hcm1.In contrast, Srx1 is dispensable for virulence in C. deuterogattii.Overall, our data demonstrate that Hcm1 is a critical determinant, which contributes to the regulatory differences of pathogenicity in C. neoformans and C. deuterogattii.

RESULTS
Hcm1 is not involved in cell cycle regulation in pathogenic Cryptococcus species CNAG_03116 is a forkhead transcription factor that is highly conserved in species belonging to Cryptococcaceae (Figure 1A), and this protein is named Hcm1 because it shares a modest similarity with Hcm1 from Saccharomyces cerevisiae, where it is a key regulator of cell cycle progression [29][30][31][32][33] .However, the conserved sequence shared between CNAG_03116 and S. cerevisiae Hcm1 is only within the forkhead domain and CNAG_03116 does not contain phosphorylation sites outside the forkhead region, which is critical for the activity of S. cerevisiae Hcm1 in cell cycle control 30,34 .These data raise the possibility that CNAG_03116 may not be important for cell cycle regulation in C. neoformans.To test this idea, we deleted its coding gene in the C. neoformans clinical reference strain (H99).In S. cerevisiae, the hcm1Δ mutant exhibited a delayed G2 phase 33 , which resulted in growth retardation.However, we found that deletion of the CNAG_03116 coding gene did not affect the growth or proliferation of C. neoformans (Figure 1B,C), consistent with the findings reported by Jung et al 11 .Additionally, phenotypic assessment combined with flow cytometry analysis indicated that the absence of CNAG_03116 in C. neoformans neither led to G2 phase delay nor caused cell size alterations (Figure 1D,E).These results demonstrate the independence of CNAG_03116 in cell cycle regulation in C. neoformans, at least under the conditions we tested.Furthermore, we further knocked out the gene encoding the homolog of CNAG_03116 in the C. deuterogattii clinical reference strain (R265) using the transient CRISPR-Cas9 coupled with electroporation (TRACE) system 35 .Gene knockout did not alter cell growth, cell size, and cell cycle progression (Figure 1B-E).These results illustrate that, unlike Hcm1 in S. cerevisiae, its orthologs in Cryptococcus pathogens are not required for cell cycle progression.Despite this, to avoid unnecessary confusion, we still used the original name of the gene (HCM1) in this study.

Hcm1 mediates the regulatory differences of pathogenicity in different Cryptococcus species
To investigate the impact of Hcm1 on pathogenicity in different Cryptococcus pathogens, we took advantage of the intranasal mouse model that mimics human cryptococcal disease 8 .Mortality curves revealed that the two independent hcm1Δ mutants generated from H99 displayed hypovirulent profiles (Figure 2A).In agreement, the rates of weight loss of mice infected by these mutants were significantly slower than that of mice inoculated with the parent strain (Figure 2C).Besides, we found that the fungal burdens in the lungs and brains of mice infected with the hcm1Δ mutant were significantly lower than those inoculated with the H99 wild-type strain (Figure 2E).Moreover, morphological observations and pathological tissue section analyses of lungs at 14 days postinoculation (DPI) showed that mice infected with the hcm1Δ mutant strain caused less damage and immune cell infiltration than the mice infected with the H99 wild-type strain (Figure S1A,B).
These data indicate that Hcm1 facilitates pathogenicity in the C. neoformans H99.Interestingly, unlike in the clinical reference strain of C. neoformans H99, deletion of HCM1 in R265 did not cause significant changes in mortality rates (Figure 2B).Consistently, similar weight loss rates and fungal burdens were observed in mice infected with the R265 wild-type and the corresponding hcm1Δ mutant strains (Figure 2D,F).Thus, Hcm1 is dispensable for virulence in C. deuterogattii R265. .(E) Fluorescence-activated cell sorting (FACS) analysis of propidium iodide-stained wild-type and hcm1Δ mutant cells cultured in YPD liquid medium for 6, 12, and 24 h.n.s, not significant.YPD, yeast extract-peptone-dextrose.

Hcm1 contributes differentially to oxidative stress defense in different Cryptococcus pathogens
To understand why Hcm1 contributes differentially to virulence in H99 and R265, we performed comparative phenotypic analysis.To this end, H99 and R265 together with their corresponding hcm1Δ mutants (two independent mutants were applied for each pathogen) were examined for their phenotypic traits under 24 distinct growth conditions.The phenotypic assessment showed that the H99-derived hcm1Δ mutants produced phenotypes that were highly similar to those reported by Jung et al. 11 , indicating the reliability of our phenotypic analysis (Figures 3A and S2A).We noticed that loss of Hcm1 did not impair the major Cryptococcus virulence factors in H99, including adaptation to host temperature (37°C) as well as production of melanin and capsule (Figure S3A-C), suggesting that these pathogenicity traits are independent of Hcm1-mediated virulence in H99.For R265, hcm1Δ likewise displayed an undetectable change in phenotypic traits in growth at 37°C or melanin synthesis but had a modestly reduced capsule thickness (Figure S3A-C).Considering that Hcm1 is dispensable for pathogenicity in R265, such reduction may be insufficient to affect the outcome of infection.These data suggest that the differences in Hcm1-mediated pathogenicity are unlikely to be due to these well-recognized Cryptococcus virulence factors.On the basis of our phenotypic evaluations, hcm1Δ mutants from H99 or R265 exhibited similar profiles of phenotypic traits, except for capsule formation as well as resistance to hydrogen peroxide (H 2 O 2 ) and 5-flucytosine (Figure 3B).We focused on the phenotype associated with H 2 O 2 tolerance, which has been well established to be critically linked with pathogenicity in various fungal pathogens 36,37 .As shown in Figure 3C, deletion of HCM1 in H99 drastically attenuated growth fitness upon exposure to H 2 O 2 , whereas there was nearly indistinguishable growth difference between R265 and its corresponding hcm1Δ mutant strains under treatment with H 2 O 2 at the same concentration.These findings demonstrate that Hcm1 homologs from H99 or R265 are distinct in their contribution to oxidative stress defense, which is important for fungal survival within host immune cells 36,38 .

Hcm1 promotes antioxidation and virulence through its direct control of sulfiredoxin Srx1 in C. neoformans
To reveal the mechanism underlying Hcm1-mediated defense against oxidative stress in C. neoformans, we performed a comparative transcriptome analysis via high-coverage RNA sequencing (RNA-seq), which targets the hcm1Δ mutant and its parent strain H99.We analyzed 6892 genes predicted to encode proteins, and thus our analysis covered 98.8% of the protein-coding genes in the entire genome.Among these genes, 164 differentially expressed genes (DEGs) were identified in response to Hcm1 deficiency, including 42 and 122 genes, downregulated or upregulated by Hcm1 (|log 2 (fold change)| > 1.0, q < 0.05) (Figure 4A).Notably, we did not detect enrichment of cell cycle-related genes in Hcm1 regulon, again supporting the conclusion that Hcm1 is not important for cell cycle progression in C. neoformans.
To evaluate the importance of the targets of Hcm1 in oxidative stress defense, we reanalyzed the publicly available transcriptomic data generated by Cheon et al., which aimed to identify DEGs of C. neoformans in response to H 2 O 2 39 .It was shown that the genes induced in response to H 2 O 2 are significantly overrepresented in the Hcm1-induced targets (Figure 4B).These results again support the importance of Hcm1 in the oxidative stress response in C. neoformans.We noticed that among the targets of Hcm1, the sulfiredoxincoding gene SRX1 displayed highly induced and abundant transcription in the presence of H 2 O 2 (Figure 4C).In fungi, Srx1 homologs generally play important roles in the recycling of hyperoxidized form (R-SO 2 H) of peroxiredoxins, which reduce inorganic and organic peroxides using electrons donated by reduced thioredoxin [40][41][42] .Upadhya et al. have demonstrated that in H99, the srx1Δ mutant was hypersensitive to H 2 O 2 .In addition, their animal experiments demonstrated that the knockout of SRX1 in H99 significantly weakened pathogenicity 43 .These phenotypes were highly similar to those observed in the hcm1Δ mutant strains.These findings led us to ask whether SRX1 acts as a key target of Hcm1 in oxidative stress defense and virulence in C. neoformans.
To test this idea, we first performed reverse transcription polymerase chain reaction (RT-PCR)-based transcriptional analyses, which confirmed the transcriptomic result, indicating the importance of Hcm1 in activating the expression of SRX1 in H99 (Figure 4D).A ChIP-qPCR analysis using an anti-FLAG antibody further revealed a significant enrichment of FLAG-labeled Hcm1 at the promoter of SRX1 (Figure 4E), suggesting that Hcm1 controls the expression of SRX1 in a direct manner.Moreover, we performed epistasis analysis for determining the genetic relationship between HCM1 and SRX1 in terms of antioxidation and virulence in C. neoformans.To achieve this, we constructed an H99-derived hcm1Δ/srx1Δ double mutant strain and overexpressed SRX1 and HCM1 in the hcm1Δ mutant and srx1Δ mutant, respectively.It was shown that the hcm1Δ/srx1Δ double mutant displayed a defective growth fitness in the presence of H 2 O 2 at a level similar to srx1Δ, which was more sensitive to oxidative stress than hcm1Δ.In addition, overexpression of SRX1 restored the defect of hcm1Δ in defense against oxidative stress, but not vice versa (Figure 4F).Significantly, the defect of pathogenicity in hcm1Δ was partially rescued when SRX1 was overexpressed in hcm1Δ (Figure 5A,D).Taken together, these results reveal that Hcm1 orchestrates oxidative stress defense and pathogenicity in C. neoformans largely through its direct target SRX1.
HCM1 plays a conserved and important role in antioxidation in different clinical strains of C. neoformans but not in those of C. deuterogattii Next, we asked whether Hcm1 likewise activates the expression of SRX1 in R265.As shown in Figure 5B, a significantly attenuated expression of SRX1 was observed in the cells where Hcm1 was absent.This finding indicates that SRX1 is a conserved target of Hcm1 in both H99 and R265.To test the impact of Srx1 on the oxidative stress response and virulence in R265, SRX1 was knocked out in this background.We first examined two independent srx1Δ mutants for their abilities to cause infections.We found that unlike srx1Δ from H99 as reported by Upadhya et al., 43 R265derived srx1Δ mutants were as virulent as their parent strain (Figure 5C), indicative of an obvious difference in the contribution of Srx1 to pathogenicity in different Cryptococcus pathogens.Consistently, a similar fungal burden in the lungs and brains was observed in mice infected with R265 wildtype and the corresponding srx1Δ mutant strains (Figure 5E).
We further evaluated the function of Srx1 in cellular tolerance to H 2 O 2 in R265 and found that although the deletion of SRX1 also reduced cellular tolerance to H 2 O 2 , the reduction was much lower than that caused by the corresponding mutation in H99 (Figure 5F).Interestingly, our phenotypic assays indicated a much stronger tolerance to H 2 O 2 of R265 relative to H99 (Figure 5F), and this finding is consistent with the results reported by Ueno et al 44 .Moreover, the resistance of H99 to H 2 O 2 was relatively similar to that of the R265-derived srx1Δ mutant (Figure 5F).Next, we asked if the observed difference in oxidative stress tolerance is strain-specific or species-specific.Thirty-eight clinically isolated strains of C. neoformans (n = 19) and C. deuterogattii (n = 19) were individually tested for their adaptation to H 2 O 2 .In this experiment, C. deuterogattii strains generally exhibited better resistance to H 2 O 2 than C. neoformans isolates (Figures 6A and S4A,B).This result supports the notion that C. deuterogattii species is more tolerant to oxidative stress than C. neoformans species, which may reflect the genetic redundancy of C. deuterogattii in antioxidation.
To test this idea, we knocked out HCM1 in different clinical strains of C. neoformans and C. deuterogattii.The corresponding mutants were subjected to phenotypic assays for evaluating their growth fitness in the presence of H 2 O 2 or during infections.Similar to the observation in H99, all C. neoformans-derived hcm1Δ mutants showed severe growth defects in the presence of H 2 O 2 (Figure 6B).By comparison, in the presence of H 2 O 2 at the same concentration, C. deuterogattii strains in which HCM1 was deleted showed largely unchanged growth (Figure 6B).We further tested the role of Hcm1 in virulence in different C. neoformans and C. deuterogattii strains.For C. neoformans strains, the lung fungal burdens in mice infected with the hcm1Δ mutants were significantly lower than those inoculated with their parent strains (Figure 6C).In contrast, mutating HCM1 in C. deuterogattii strains led to modest or undetectable impairment of cryptococcal survival in the lungs (Figure 6D).Altogether, these

DISCUSSION
Despite a close evolutionary relationship, C. neoformans and C. deuterogattii have disparate clinical characteristics 4 .C. neoformans primarily infects immunodeficient populations, such as AIDS patients, and often cause central nervous system infections 4,45 .However, C. deuterogattii is frequently associated with infections in immunocompetent populations and can cause severe lung disease without dissemination to other organs 46,47 .These dissimilarities indicate that these pathogens may employ different molecular machines and regulatory systems for enabling their pathogenicity.An earlier study performed by Lam et al. demonstrated that the chitin deacetylase Cda1 is essential for virulence in C. neoformans KN99, while in C. deuterogattii R265, Cda3 rather than Cda1 is the major chitin deacetylase engaged in pathogenicity 48 .Most recently, Lee et al. showed that the homeobox transcription factor Hob1 facilitates brain infection in H99, but its homolog in R265 does not 49 .Despite these important findings, the determinants and mechanisms underlying the interspecies regulatory differences of pathogenicity remain unclear.
Our data indicated that the regulatory differences are partially attributed to the forkhead transcription factor Hcm1. Due to sharing a modest similarity with the cell cycle regulator Hcm1 from the ascomycete S. cerevisiae, this regulator was considered to play a similar role in cell cycle control in the basidiomycete C. neoformans.However, results of our phenotypic and transcriptomic analyses showed that Hcm1 does not play role in the regulation of the cell cycle in either C. neoformans or C. deuterogattii, indicating a disparity of function between Hcm1 orthologs from S. cerevisiae and Cryptococcus pathogens, which diverged 500 million years ago 50 .We further revealed the importance of Hcm1 in antioxidation in C. neoformans.The Hcm1-directed orchestration of the oxidative stress response and pathogenicity in this pathogen largely depends on its direct regulation of sulfiredoxin-coding gene SRX1, which is dedicated to reducing inorganic and organic peroxides through recycling of peroxiredoxins in various fungi, including C. neoformans 40,41 .Interestingly, although the regulatory relationship between Hcm1 and SRX1 was also identified in C. deuterogattii R265, neither of them is required for virulence in this pathogen.Phenotypic analyses revealed that the contribution of Hcm1 and Srx1 to oxidative stress defense in R265 is obviously weaker than their homologs in H99.Of note, R265 showed a much stronger tolerance to hydrogen peroxide than H99.This finding is consistent with the previous observation that R265 has a better fitness in macrophages than H99 due to its more potent ROS tolerance [26][27][28] .We further revealed that the distinction in ROS defense is not strain-specific but speciesdependent.By monitoring the fitness of dozens of clinical isolates from two species, it was found that C. deuterogattii strains generally are robust in ROS tolerance compared with isolates of C. neoformans.This suggests the possibility that the genetic redundancy of antioxidation in C. deuterogattii may be related to a species-specific feature regarding strong ROS tolerance.Supporting this, the wild-type-like antioxidant capability was detected upon deletion of HCM1 in C. deuterogattii clinical strains in the presence of high H 2 O 2 , which otherwise resulted in a dramatic reduction in growth fitness in hcm1Δ mutants generated from C. neoformans strains.
Clinically, C. deuterogattii is considered to be more virulent than C. neoformans in terms of lung infection [51][52][53] .It has been well recognized that during lung infection, the survival of pathogens is highly associated with their abilities to defend against ROS released by immune cells, such as macrophages 36,38 .In this regard, the better adaptation to ROS by C. deuterogattii may partially explain its excellent capability for enabling lung infection.Further dissection of speciesspecific gene networks underlying antioxidation in these two Cryptococcus species may provide important insights into how their pathogenicity features during lung infections are specified and may indicate novel targets for the development of antifungals against these two important fungal pathogens.

Strains and growth conditions
The strains used in this study are listed in Table S1.C. neoformans and C. deuterogattii were cultured on yeast extract-peptone-dextrose (YPD) liquid medium (1% yeast extract, 2% Bacto peptone, 2% dextrose, and 2% Bacto agar) at 30°C unless specified otherwise.Dulbecco's modified Eagle's (DME) medium was used to measure capsule production at 37°C in 5% CO 2 54 .Minimal medium (MM) containing L-dihydroxyphenylalanine (L-DOPA) was used to test melanin production at 30°C in the dark 55 .

Gene disruption and overexpression
The primers used in this study are listed in Table S2.Targeted gene deletion was performed as previously described 56 .Briefly, overlapping PCR products were generated with nourseothricin (NAT) or neomycin (NEO) resistance cassettes and 5′ and 3′ flanking sequences (1.0-1.5 kb) of the target genes from C. neoformans and C. deuterogattii strains as previously described.The PCR products were introduced into relevant recipient strains by the TRACE method 35 .Genotypic accuracy of the positive transformants was confirmed by PCR.For gene overexpression, genes (open reading frame-ORF) were amplified by PCR and were cloned into the plasimd pXC 56 with a FLAG tag behind the P CnH3 promoter and introduced into the safe haven regions 57 of relevant recipient strains by electroporation.

Growth curve assay
Cells were inoculated into culture tubes containing YPD liquid medium at a starting cell density of A 600 = 0.1 (A 600 : absorbance at 600 nm).Cells were incubated at 30°C and 37°C with shaking at the rate of 220 rpm.The cell concentration was measured at the indicated time points with an ultraviolet-visible spectrophotometer.

Phylogenetic analysis
The Hcm1 protein sequence from C. neoformans H99 was used as the query sequence for Basic Local Alignment Search Tool (BLAST) search.The phylogenetic trees for Hcm1 homologs were developed with their whole-protein sequences by using the neighbor-joining method in MEGA-X.

Flow cytometry analysis
Analysis of the Cryptococcus cell cycle by flow cytometry was conducted according to a method described previously 58,59

Growth and chemical susceptibility analyses
Growth and chemical susceptibility analyses were performed as described previously 11 .Briefly, cells were cultured overnight in YPD liquid medium at 30°C with shaking at 220 rpm, and then washed twice with PBS, adjusted to the same cell density (5.0 × 10 7 CFU/ml), and were five-fold serially diluted.
Cells were incubated at 30°C in the dark for 2-3 days and photographed.

Assays for capsule and melanin production
Capsule production was measured as described previously 54 .Briefly, cells were cultured overnight in YPD liquid medium at 30°C with shaking at 220 rpm, then washed three times with PBS and diluted to a final concentration of 1.0 × 10 5 /ml in a DME medium.To induce capsule formation, the cells were incubated at 37°C with 5% CO 2 for 72 h.The capsules were visualized by negative staining with India ink under a light microscope.To analyze melanization, cells were cultured overnight in YPD liquid medium at 30°C.The cells were washed twice with PBS and then adjusted to the same cell density (5.0 × 10 7 CFU/ml) and fivefold serially diluted.The diluted cells were spotted on MM containing L-DOPA, incubated at 30°C for 3 days in the dark, and photographed 55 .

Murine model of cryptococcosis
Mouse lung infections were performed as previously described 8,60 .Briefly, female C57 mice (7-8 weeks old) were subjected to cryptococcal infection by inhalation.Cryptococcal strains were grown in YPD liquid medium overnight at 30°C with shaking at 220 rpm.The fungal cells were washed twice with PBS and adjusted to 2.0 × 10 6 CFU/ml.For survival assays, mice (n = 10) were randomly distributed into each group.The mice were anesthetized with ketamine and xylazine and then infected with 50 μl of fungal cell suspensions via intranasal instillation.After infection, animals were observed daily for disease progression, including weight changes and labored breaths.

Organ fungal burden analysis
For organ fungal burden analysis, the infection procedure was the same as for the survival assay.The lungs and brains of three infected mice at DPI 14 were homogenized using a homogenizer and spread on a YPD solid medium containing 100 μg/ml of chloramphenicol.The plates were incubated at 30°C for 2-3 days, and then CFUs were counted manually.

Histology assays
Histology assays were performed as described previously 61,62 .Briefly, mice infected with different Cryptococcus strains were killed at DPI 14.The lungs were harvested and fixed in 10% formalin solution and sent to the Peking Union Medical College Hospital for section preparation.Tissue slides were stained with hematoxylin and eosin (H&E) staining and examined by light microscopy.

RNA-seq and data analysis
For the RNA-seq analysis, the wild-type H99 and its corresponding hcm1Δ mutant strain were cultured in a YPD liquid medium at 30°C for 6 h.RNA extraction was performed as previously described 56,63,64 .Briefly, total RNA was extracted using Ultrapure RNA Kit (CW0581M, CWBIO) according to the manufacturer's instructions.RNA-seq and data analysis were performed as previously described.Briefly, RNA-seq was performed by Annoroad Gene Technology Co., Ltd.Initial quality control of sequenced clean data was performed using FastQC v0.

Quantitative RT-PCR analysis
To test whether SRX1 is regulated by Hcm1 both in C. neoformans H99 and C. deuterogattii R265 backgrounds.H99 and R265 wild-type and corresponding hcm1Δ mutants were cultured in YPD liquid medium overnight at 30°C with shaking at 220 rpm and then transferred to RPMI 1640 medium at 37°C with 5% CO 2 to mimic host physiological environment.The cells were collected at 6 h postincubation for the isolation of total RNA.Total RNA was extracted using an Ultrapure RNA Kit (CW0581M, CWBIO) according to the manufacturer's instructions.The RNA was reversetranscribed with the Fastquant RT Kit (with gDNase, KR106-02, Tiangen).The relative mRNA level of SRX1 was calculated by real-time RT-PCR.The relative transcript levels of SRX1 were normalized to that of the constitutively expressed housekeeping gene TEF1 and determined by the comparative Ct method as described previously 54 .

ChIP-assay
ChIP-assays were performed as described previously [65][66][67] .Briefly, cells were cultured in RPMI 1640 medium for 6 h in an incubator containing 5% CO 2 at 37°C.Cells were then harvested and cross-linked with 1% formaldehyde.Glycine (final concentration 125 mM) was subsequently added to quench the cross-linking reaction.Micrococcal nuclease was used to fragment chromatin to an appropriate size.Clarified chromatin extracts were immunoprecipitated with an anti-FLAG antibody.The DNA-protein complex was then washed and eluted, and eluted samples were subjected to reversal of cross-linking.Afterward, DNA was purified by the phenol-chloroform extraction method and precipitated in ethanol.Primers upstream of the SRX1 ORF were used to perform qPCR.The ChIP enrichment signal was quantified as the percentage of input.Primers used in ChIP-qPCR analyses are listed in Table S2.

Statistical analysis
Statistical analyses were performed with Prism 8.0.We used two-tailed Student's t tests to compare lung and brain fungal burden, cell size, survival index, or transcript levels from two groups.Gehan-Breslow-Wilcoxon tests were utilized to evaluate the significance of survival data for the various groups.For all analyses, p value less than 0.05 (typically <0.05) is statistically significant and p value higher than 0.05 (>0.05) is not statistically significant.In all figures, the data are shown as mean ± standard deviation (SD) from three or more independent biological replicates.

Figure 1 .
Figure 1.Hcm1 is not involved in cell cycle regulation in Cryptococcus pathogens.(A) Phylogenetic analysis of Hcm1 homologs.The phylogenetic tree was generated with MEGA-X using the neighbor-joining method.The bar marker indicates the genetic distance proportional to the number of amino acid substitutions.Bootstrap values based on 1000 replications are indicated at the branch points.Growth curves (B) and budding index (C) of the wild-type and hcm1Δ mutants in the Cryptococcus neoformans H99 and Cryptococcus deuterogattii R265 background.(D) Violin plots showing the cell size distribution of wild-type and hcm1Δ mutant strains.Cryptococcus cells were grown in YPD liquid medium for 6, 12, and 24 h (n > 50).(E) Fluorescence-activated cell sorting (FACS) analysis of propidium iodide-stained wild-type and hcm1Δ mutant cells cultured in YPD liquid medium for 6, 12, and 24 h.n.s, not significant.YPD, yeast extract-peptone-dextrose.

Figure 2 .
Figure 2. Hcm1 promotes pathogenicity in Cryptococcus neoformans but not in Cryptococcus deuterogattii.Ten C57 BL/6 female mice were infected with wild-type and hcm1Δ mutant strains in C. neoformans H99 (A) and C. deuterogattii R265 (B) backgrounds.The survival percentage of infected mice and uninfected mice was monitored over 60 days postinfection.Dynamic curves of the body weight changes in mice infected with wild-type and hcm1Δ mutant strains in C. neoformans H99 (C) and C. deuterogattii R265 (D) backgrounds, respectively.C57 BL/6 female mice were infected with the wild-type and hcm1Δ mutant strains in C. neoformans H99 (E) and C. deuterogattii R265 (F) backgrounds.Fungal burden in the lungs and the brains was assessed by counting CFU at DPI 14.Data represent the mean ± SD (n = 3).***p < 0.001, and n.s, not significant.CFU, colony forming unit; DPI, days postinoculation; PBS, phosphate-buffered saline; SD, standard deviation.

Figure 3 .
Figure 3. Hcm1 contributes more strongly to oxidative stress defense in Cryptococcus neoformans H99 than in Cryptococcus deuterogattii R265.(A) Semiquantitatively phenotypic assays of hcm1Δ mutants constructed in C. neoformans H99 and C. deuterogattii R265 backgrounds based on data presented in Figure S2.Red and yellow circles represent defective and enhanced, respectively.The gradients of red or yellow indicate the phenotype strengths (strong, intermediate, and weak).(B) Phenotypic comparison of the hcm1Δ mutants constructed in C. neoformans H99 and C. deuterogattii R265 backgrounds, based on data presented in Figure S2.(C) Cells were grown overnight in YPD at 30°C, five-fold serially diluted, spotted onto YPD medium containing H 2 O 2 at the indicated concentrations, and incubated for 2-3 days before photographing.

Figure 4 .
Figure 4. Hcm1 promotes antioxidation through its direct control of the sulfiredoxin Srx1 in Cryptococcus neoformans.(A) Volcano diagram of the differentially expressed genes in comparison with the wild-type and hcm1Δ mutant strain in the C. neoformans H99 background.(B) Gene Set Enrichment Analysis demonstrated that enrichment of H 2 O 2 -induced genes in the targets was transcriptionally activated by C. neoformans Hcm1.(C) Transcriptional induction response of Hcm1 targets to hydrogen peroxide.The red dot indicates the sulfiredoxin-coding gene SRX1.The original transcriptome data for DEGs in response to hydrogen peroxide were obtained from a previous study 39 .(D) RT-PCR analysis of SRX1 transcription in different strains from H99 background.Data represent the mean ± SD (n = 3).(E) Chromatin immunoprecipitation (ChIP) was performed with an anti-FLAG antibody in hcm1Δ cells in which the FLAG-fused Hcm1 was overexpressed.ChIP enrichment was detected by qPCR across an approximately 220 bp region upstream of the SRX1 ORF.Data represent the mean ± SD (n = 3).(F) Spotting susceptibility assays of wild-type, hcm1Δ, srx1Δ, hcm1Δ/srx1Δ, hcm1Δ/P CnH3 -HCM1, srx1Δ/P CnH3 -SRX1, hcm1Δ/P CnH3 -SRX1, and srx1Δ/P CnH3 -HCM1 strains in the C. neoformans H99 background were performed in the presence of H 2 O 2 at the indicated concentration.*p < 0.05, **p < 0.01.DEGs, differentially expressed genes; FC, 5-flucytosine; FDR, false discovery rate; NES, normalized enrichment score; ORF, open reading frame; qPCR, quantitative polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction.

Figure 5 .
Figure 5. SRX1 as a downstream target of Hcm1 promotes virulence in H99 but not in R265.(A) Survival curves plotted against time upon challenge with wild-type, hcm1Δ, hcm1Δ/P CnH3 -HCM1, and hcm1Δ/P CnH3 -SRX1 strains in the Cryptococcus neoformans H99 background by intranasal instillation.(B) RT-PCR analysis of SRX1 transcription in wild-type and hcm1Δ strains in the Cryptococcus deuterogattii R265 background.(C) Survival curves plotted against time upon challenge with wild-type and srx1Δ strains in the C. deuterogattii R265 background by intranasal instillation.(D) Lung and brain fungal burdens from mice that were infected with wild-type, hcm1Δ, hcm1Δ/P CnH3 -HCM1 and hcm1Δ/P CnH3 -SRX1 in the C. neoformans H99 background at DPI 14. (E) Lung and brain fungal burdens from the mice that were infected with wild-type and srx1Δ strains in the C. deuterogattii R265 background at DPI 14. (F) Spotting susceptibility assays of wild-type, hcm1Δ, and srx1Δ strains in H99 and R265 backgrounds were performed in the presence of H 2 O 2 at the indicated concentration.Data represent the mean ± SD (n = 3).**p < 0.01, ***p < 0.001, and n.s, not significant.

Figure 6 .
Figure 6.HCM1 plays a conserved and important role in antioxidation and pathogenicity in different clinical strains of Cryptococcus neoformans but not in those of Cryptococcus deuterogattii.(A) Semiquantitatively phenotypic assays were performed in the presence of H 2 O 2 at the indicated concentration.The survival index of C. neoformans strains (n = 19) and C. deuterogattii strains (n = 19) were based on data presented in Figure S4.(B) Spotting susceptibility assays of hcm1Δ mutants derived from different C. neoformans and C. deuterogattii strains and their corresponding parent strain were performed in the presence of H 2 O 2 at the indicated concentration.Data represent the mean ± SD (n = 3).(C) Lung fungal burdens from the mice infected with different C. neoformans strains and their corresponding hcm1Δ mutants at DPI 14. (D) Lung fungal burdens from the mice infected with different C. deuterogattii strains and their corresponding hcm1Δ mutants at DPI 14.Data represent the mean ± SD (n = 3).***p < 0.001.
11.5 software.Then, Hisat2 v2.1.0was used for clean short reads mapping to the genome sequence of C. neoformans H99.The relative transcript abundances were measured in transcripts per million by Stingtie v1.3.3 to determine unigenes.DEGs were identified with DEseq. 2 and defined based on the basis of the fold change criterion (|log 2 (fold change)| > 1.0, q < 0.05).
. Briefly, Cryptococcus strains were collected at different time points (i.e., 6, 12, and 24 h) after transferring the overnight cultures to fresh YPD liquid medium, then washed twice in phosphate-buffered saline (PBS) and fixed in 70% ethanol overnight at 4°C.Fixed cells were washed once with NS buffer (10 mM Tris-HCl [pH 7.6], 250 mM sucrose, 1 mM ethylenediaminetetraacetic acid [pH 8.0], 1 mM MgCl 2 , 0.1 mM CaCl 2 , 0.1 mM ZnCl 2 , 0.4 mM phenylmethylsulfonyl fluoride, and 7 mM β-mercaptoethanol).Cells were stained with propidium iodide (12.5 μg/ml) in NS buffer containing RNase A (1.0 mg/ml).The mixture was incubated with agitation at room temperature for 2-3 h.Stained cells were diluted into Tris-HCl (pH = 8.0) buffer and sonicated for 1 min.Finally, 10,000 cells were analyzed on the FL2 channel on a BD FACSCalibur at the Beijing Regional Center of Life Science Instrument, Chinese Academy of Sciences.